Mycobiota of rape seeds in Romania . I . Identification of mycobiota associated with rape seeds from different areas of Romania

The spectrum of fungal diversity associated with rape seeds belonging to 33 cultivars (Alaska, Astrada, Astrid, Atlantic, Betty, Champlein, Chayenne, Dexter, Digger, Elvis, Eurowest, Finesse, Herkules, Hydromel, Hydromel-MA, Ladoga, Manitoba, Masa Rom, Milena, Mohican, Montego, Nectar, Ontario, Orkan, Perla (4 lots), Remy, Robust, Rodeo, Saphir, Tiger, Tiger CBC Lot ROM06-121-110, Triangle, Valesca, Vectra) and 2 hybrids (H90-20-83, H-90-21-83) has been established by samples’ macroscopical and microscopical analizying, during 2006-2008, for the first time in Romania. The Ulster method on malt-agar and PDA culture media has been used, evaluating the percentage of fungal taxons present on/ in rape seeds. The most important pathogenic fungi identified were: Sclerotinia sclerotiorum (Lib.) de Bary, Botrytis cinerea Pers., Rhizoctonia solani Kühn, Alternaria brassicae (Berk.) Sacc., A. brassicicola (Schwein.) Wiltshire and Fusarium spp. Also, a large quantities of some saprophytic fungi, as Alternaria, Cladosporium, Aspergillus, Penicillium, Rhizopus have been recorded. These ones have been affected the health condition of rape seeds, suppressing their germination and other vital phenomena. Among potential antagonistic fungi the followiung genera have been isolated: Chaetomium (0-4%), Trichoderma (0-10%), Aspergillus (0-14%), Penicillium (0-100%). Some correlations and comparisons have been established between fungal diversity, their provenience, cultivars, culture media (Malt-Agar/MA, Potato-DextroseAgar/PDA) used. It has been evaluated the behaviour of rape cultivars and hybrids towards the main rape seed pathogens.


INTRODUCTION
Rape is in Romania at present an extending crop in comparaison with the years 2001-2005, when its area was in a significant decreasing.The largest area of rape crop has been recorded until now in 2010, the increasing of its surface starting since 2007 (Tab.1).The highest values of the rape yields (569.6-943.0thousands tones/ha) have been obtained during 2008-2010 and the lowest one (8.1-98.7 thousands tones/ha) during 2002-2004.Regarding the average yield evaluated in kg rape seeds/ha, the highest seed production (1984 kg/ha) was recorded in 2004, followed by the values of 1357-1844 kg/ ha between 2005-2010, with an exception (991 kg/ha) in 2007.In order to obtain a constant valuable rape yield it has to be applied in the crop technology the suitable agrotechnological and protection methods, providing a healthy crop developped from high quality seeds, free of seed-borne pathogens, with a high germinative faculty.Also, in the frame of the sustainable agriculture concept, it has to be substitute, as much as possible, the chemicals with the ecological, non-chemical products or with different other alternative non-polluting protection means (Baicu, Săvescu 1986;Hălmăjan et al. 2006).These objectives have been followed during our researches, starting with an approach on endo-and epiphytic mycobiota of rape seeds belonging to various cultivars from different pedoclimatic zones of Romania.
In general, there are a few researches on seed pathology in Romania, like books of Hulea et al. (1973) or Raicu and Baciu (1978).Also, no papers referring to the mycobiota of rape seeds in Romania have been presented, but not published in extenso until now (Tabuc, Ştefan 2003;Şesan, Groza 2007;Şesan 2008).This paper is the first published contribution in the field of seed-pathology of rape in order to have an evaluation of rape seed-borne fungi/mycobiota as a reference point for obtaining a healthy and efficient, productive rape crop.
The objective of this study has been the investigation and evaluation the rape seed healthiness and detection of potential active antagonistic fungi against the main rape phytopathogens in order to be used in the future in their biocontrol.
Methods used for the identification of mycobiota associated with rape seeds were: macroscopical analyze, optical microscopy (microscope Olympus CX 41 from Japan, with a digital soft camera control panel SP-350), wet chamber and Ulster methods, on two culture media: Malt-Agar (MA) and Potato -Dextrose -Agar (PDA) (Neergaard 1979;Musket, Malone 1997;Machado et al. 2002).
The evaluation of the data has been done by the frequency (F) of seed-and soilborne fungi associated with the rape seed samples (Tab.2, Figs 1-3).

RESULTS AND DISCUSSION
The performed results on identification of mycobiota associated with rape seeds from different areas of Romania are presented in the Table 2, Figs. 1-3 and Plates 1-5.
Among these, the first significant phytopathogens (S. sclerotiorum, B. cinerea) haven't been identified in all analyzed seed samples from the yields of 2006-2007 years.Absence of these seed-borne fungi was correlated with the crop rotation, avoiding sensitive plants (sunflower, soybean, carot, potato) to these pathogens in the same asolament and especially with the very dry climatic conditions during 2006-2007.
It has been identified in only one sample, cv.Eurowest, the fungus Rhizoctonia solani Kühn, with a low frequency of 1%.
Among soil-borne pathogens, there were identified Fusarium spp., with a high frequency until 51%, and average limits between 0.4 until 10.25%, present in 62.5% of seed samples.The highest level of Fusarium spp.has been detected on the Masa Rom cv.(51%), followed by Hydromel-MA cv.(26%), Elvis (22%) and then by the hybrid H-90-21-83 (12%).The lowest occurence of Fusarium spp.(F = 1%) has been detected for the cultivars: Tiger, Dexter, Finesse and Herkules.In the performed investigations, Rhizoctonia solani have been identified on MA culture medium and it has not been recorded in our tests on the PDA culture medium.Also, in the Petri plates, on PDA medium, Trichoderma spp.have been identified.On the same culture medium (PDA) yeasts and bacteria associated with rape seeds presented a good development (Tab.2, I-II) in comparaison with MA medium.
These results explain why ISTA recommends Ulster method on the MA culture medium as a generalized standard method for seed pathology/ phytosanitary tests for the majority of cultivated plants (Malone, Muskett 1997;Machado et al. 2002).

A strong contamination of rape seed samples from the South of Romania with
Rhizopus stolonifer represents a risk factor in obtaining a healthy and highly productive rape crop. 5.Among the potential antagonistic fungi associated with rape seeds the following species have been detected: Chaetomium spp.(0-4%), Trichoderma spp.(0-10%), Aspergillus (0-14%), Penicillium (0-100%) a.o.This category of micromyceta are important in the selection of actives isolates for using them in biocontrol of phytopathogens.
On the other hand, some cultivars proved sensitivity to the main seed-and soilborne fungi: 3 sensitive cultivars (Masa Rom., Hydromel-MA, Elvis) to Fusarium spp., 4 cultivars (Milena, Perla Lot 6B00751-6B003, Alaska, Robust) and 1 hybrid (H-90-21-83) to Alternaria spp., and 6 cultivars (Ontario,Nectar,Rodeo,Saphir,Triangle) to R. stolonifer.These data are very useful to the practice for selection of cultivars/hybrids in order to obtain a healthy and productive rape crop.

Table 1
Dynamics of rape crop areas and yields during 2001-2010 in Romania (after the Statistical Yearbooks of Romania, 2007-2011)

Table 2
Main fungal pathogenic and saprotrophic species associated with rape seeds in România No .